We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Method Development. The use of label-free approaches for quantitative proteomics studies has been implemented. This methodology involves data-independent analyses (MSe) and an online UPLC method suitable for the analysis of proteins from complex samples. In theory, MSe can rapidly acquire accurate mass precursor and fragment ion information while simultaneously obtaining fairly accurate quantitative profiles from every detectable component in the sample. The data obtained from depleted serum (depletion of the 14 most abundant proteins) followed by six different sample treatments has been acquired. The results obtained by data-independent analyses and by data-dependent analyses are being evaluated. Additionally, we are incorporating ion mobility methods in an effort to gain further information. Ion mobility is a useful tool to aid in mass spectrometry applications because it allows for the measurement of the collisional cross section of a molecule and gives information about the three-dimensional shape of a compound in the gas phase. Ion mobility separates ions based on their differential mobility through a buffer gas based on the ions shape, charge, and mass. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions and thus travel more slowly than those ions that comprise a smaller cross-section. Thus, ion mobility serves as a useful means of orthogonal separation in unrelated molecules as well. This is a real benefit because the ability to identify and quantify proteins is directly linked to the power of chromatographic separations. 2. Myositis Study. A differential proteomics project comparing the quantitation of proteins from sera of healthy individuals to individuals diagnosed with a rheumatic disease is underway. This work is in collaboration with F. Miller (EAG) as part of the EAGs study of families with twins or siblings discordant for systemic rheumatic disorders. The goal of this project is to determine whether a protein or a suite of proteins can be identified that would allow for the early diagnosis, prognosis, and treatment of these diseases. Sera samples have been depleted and digested, data has been acquired, and the results are being processed. 3. Tissue Study. A protein expression study of LCM tissue is underway. Frozen rat brain tissue has been evaluated for protein extraction using various extraction protocols. The best results gave an MS identity of about 300 proteins. A study comparing the extraction efficacy of proteins from PAXgene fixation versus traditional fixation is under investigation. 5. Acrylamide Study. The effects of sterilization parameters on rodent feed have been determined by measuring the amount of acrylamide that is produced in the feed with different sterilization parameters, primarily different steam autoclaving times and number of cycles. Analysis of acrylamide and its genotoxic metabolite glycidamide in rat plasma and urine is currently ongoing. Mass spectral analysis of these low mass polar analytes in complex matrices is challenging. We have developed a novel derivatization method to aid in the extraction, separation, and ionization of these target analytes in biological samples. 6. Isoprostane Study. Isoprostanes are a family of compounds that are identical to their corresponding prostaglandins except for differences in stereochemistry. Because isoprostanes can be formed nonenzymatically, they are often studied as markers of oxidative stress. The generation of the most often measured isoprostane, 8-iso-PGF2alpha; by prostaglandin-endoperoxide synthase and arachidonic acid even with low enzymatic activity has been investigated. From our studies, an increase in the concentration of 8-iso-PGF2alpha; does not necessarily reflect an increase in chemical lipid peroxidation. We recommend using the 8-iso-PGF2alpha;/PGF2alpha; ratio to quantitatively distinguish between increases in 8-iso-PGF2alpha; by enzymatic and/or chemical lipid peroxidation. 7. Lipid/Fatty Acid Studies. A) We have quantified cholesterol metabolites in human serum and lung lavage as well as samples from animal and cell culture experiments. From initial studies, a correlation between the levels of these compounds and individuals with Acute Respiratory Distress Syndrome has been observed. Additional samples are currently being interrogated to assess the usefulness of these compounds as markers of respiratory distress. B) The autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis is being investigated as a molecular target for lowering intraocular pressure (IOP) in animal models of glaucoma. Using ATX inhibitors, the role of autotaxin in regulation of intraocular pressure is characterized with the detection of LPA and LPC in the aqueous humor of eye. C) The role of COX-2 as an upstream regulator of p53 levels was investigated. DNA damage-induced increase in p53 protein levels was greatly reduced in COX-2 knockout (KO) mouse embryonic fibroblasts (MEFs) compared to that in wild type or COX-1 KO MEFs. Furthermore, a COX-2 specific inhibitor suppressed doxorubicin-induced p53 accumulation and cell death. D) The role of the retinoid acid-related orphan receptor gamma, ROR gamma, in the transcriptional regulation of lipid metabolic genes was determined by the quantitation of fatty acids. E) We are looking at a comparison of short chain fatty acids in feces to determine if obesity has an effect on the metabolism of cells. It was shown previously that obesity can result in some epigenetic changes in colon epithelial cells and make them resemble tumor cells. 8. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 49 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. 9. Buprenorphine Study. Buprenorphine is an opiate which is used to control pain in experimental animals by our CMB. Plasma concentrations of buprenorphine drop below therapeutic levels between 2 and 4 hours after subcutaneous injection. To achieve a longer therapeutic window, sustained release formulations and pluronic gel formulations are being investigated. Buprenorphine, norbuprenorphine, and their glucuronides in plasma have been quantified from initial formulations. 10. Steroid Studies. Steroid hormones are widely distributed in nature and are potent signaling molecules. As such, they are of interest to several researchers within the institute. We have done a comparison of the MS sensitivity for a variety of steroids on different instruments in our laboratory. A supported liquid extraction protocol followed by chemical derivatization of estrogens has been developed to allow the simultaneous quantification of androgens, estrogens, and the estrogenic compound diethylstilbestrol in plasma. We are currently evaluating the expansion of the methodology to include corticosteroids and bisphenols.